Characterization of circulating extracellular vesicle content and PAG concentrations in pregnant sheep infected with BVDV

The placenta is an active immunologic organ, modulating the maternal environment to support the conceptus. Fetal trophoblasts produce a variety of products including pregnancy-associated glycoproteins (PAGs) and extracellular vesicles called exosomes. PAGs directly interact with the uterus and maternal immune cells with low concentrations correlating with adverse pregnancy outcomes in cattle. Exosomes contain nucleic acids and proteins that influence intercellular communication during pregnancy. The specific objectives of this body of work were to 1) quantify circulating PAG concentrations, 2) characterize the profiles of circulating exosomal proteins and miRNAs in pregnant and non-pregnant sheep, and 2) determine the difference in exosomal content profiles between non-infected and BVDV-infected pregnant and non-pregnant sheep. Twenty-four yearling ewes, found to be negative to BVDV on serum neutralizing antibody serology, were enrolled in this study. Ewes were inoculated with BVDV NY-1 or sham media. Fifteen days post inoculation, all animals underwent hysterectomy and peripheral blood collection. PAG1 concentrations were quantified using commercially available ELISA. Exosomes were isolated using ultracentrifugation and gradient density separation. Small RNA high through-put sequencing and mass spectroscopy proteomics were performed. BVDV infection status was determined with BVDV PCR of fetal tissue, IHC of placentomes, and VN of dams. Statistical differences between circulating concentrations of PAG1, miRNAs, and proteins were analyzed by analysis of variance for repeated measures. PAG1 concentrations in maternal blood differed between treatment groups, with mean concentrations significantly lower (P =0.04) in BVDV inoculated dams compared to controls. Sixty million reads, identifying 1634 miRNAs were identified following high-throughput sequencing. Several miRNAs were identified to be unique to BVDV exposure and pregnancy status. With a confidence rate of 0.01, 539 proteins were identified with differential relative abundance between viral exposure. In conclusion, infection with BVDV decreases PAG1 concentration in maternal circulation in pregnant sheep compared to healthy controls. Exosomes containing select miRNAs and proteins are present in peripheral circulation likely have a biological role in fetal-maternal interactions important in times of both disease and health. Maternal PAG concentrations and differential exosome content may be a viable biomarker to determine placental health following reproductive viral infections.